IFI30 as a key regulator of PDL1 immunotherapy prognosis in breast cancer.

BACKGROUND: IFI30 is a lysosomal thiol reductase involved in antigen presentation and immune regulation in various cancers, including breast cancer. Despite its known involvement, the precise mechanism, function, and relationship with the PD-L1 axis and immune response remain unclear. METHODS: We conducted an extensive investigation into IFI30 mRNA expression in breast cancer utilizing data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Furthermore, we characterized IFI30 mRNA expression across various cell types using publicly available single-cell RNA sequencing datasets, and assessed protein expression through immunohistochemistry using an in-house breast cancer tissue microarray. Functional experiments were performed to elucidate the effects of IFI30 overexpression on PD-L1 expression and inhibitory efficacy in both macrophages and breast tumor cells. RESULTS: Our study unveiled a marked upregulation of IFI30 expression in breast cancer tissues compared to their normal counterparts, with notable associations identified with tumor stage and prognosis. Additionally, IFI30 expression demonstrated significant correlations with various immune-related signaling pathways, encompassing peptide antigen binding, cytokine binding, and MHC class II presentation. Notably, breast cancer samples exhibiting high IFI30 expression in tumor cells displayed high PD-L1 expression on corresponding cells, alongside a diminished ratio of CD8 + T cell infiltration within the tumor microenvironment. Furthermore, ectopic knockdown of IFI30 in both tumor cells and macrophages resulted in a reduction of PD-L1 expression, while conversely, overexpression of IFI30 led to an increase in PD-L1 expression. CONCLUSIONS: This study offers new insights into the involvement of IFI30 in breast cancer, elucidating its interplay with the PD-L1 axis and immune response dynamics. Our findings suggest that modulation of the IFI30-PD-L1 axis could serve as a promising strategy for regulating T cells infiltration in breast cancer thus treating breast cancer.

circNFIB decreases synthesis of arachidonic acid and inhibits breast tumor growth and metastasis.

We identified circNFIB (hsa_circ_0086376) as a down-regulated circRNA in breast cancer but its effect is unclear. We aimed to explore the roles of circNFIB in breast cancer. The expression levels of circNFIB in breast cancer tissues and cells were detected. Both in vitro and in vivo experiments were used to assess the effects and mechanisms of circNFIB. circNFIB was down-regulated in 29 breast cancer tissues compared to adjacent normal tissues. circNFIB is a highly conserved circRNA and mainly located in cytoplasm of breast cancer cells. In vitro experiments showed that overexpression of circNFIB inhibited proliferation and invasion of breast cancer cells, whereas knockdown of circNFIB induced proliferation and invasion. Animal experiments indicated that circNFIB inhibited tumor growth and metastasis in vivo. Bioinformatics analysis showed that circNFIB contained an open reading frame (ORF) spanning its spliced junction, an internal ribosome entry site (IRES) and a N6-methyladenosine (m6A) site, suggesting circNFIB had the potential to encode a 56 amino acid (aa) protein, which was then confirmed by experiments. Metabonomics analysis results indicated that circNFIB may inhibit synthesis of arachidonic acid (AA) by regulating phospholipase. EIF4A3 and U2AF65 may regulate circNFIB expression by binding to the flanking sequence of circNFIB. In conclusion, circNFIB is a down-regulated circRNA in breast cancer tissues and encodes a 56 aa protein. circNFIB down-regulates AA in breast cancer cells, thus decreasing AA metabolites. Based on reported evidences of AA metabolites on cancer, we speculated that circNFIB may inhibit breast tumor growth and metastasis partly by inhibiting AA.

Glucose metabolism and lncRNAs in breast cancer: Sworn friend.

BACKGROUND: Glucose metabolism disorder is a common feature in cancer. Cancer cells generate much energy through anaerobic glycolysis, which promote the development of tumors. However, long non-coding RNA may play an important role in this process. Our aim is to explore a prognostic risk model based on the glucose metabolism-related lncRNAs which provides clues that lncRNAs predict a clinical outcome through glucose metabolism in breast cancer. METHODS: 1222 RNA-seq were extracted from the TCGA database, and 74 glucose metabolism-related genes were loaded from the GSEA website. Then, 7 glucose metabolism-related lncRNAs risk score model was developed by univariate, Lasso, and multivariate regression analysis. The lncRNA risk model showed that high-risk patients predict a poor clinical outcome with high reliability (P=2.838×10-6). Univariate and multivariate independent prognostic analysis and ROC curve analysis proved that the risk score was an independent prognostic factor in breast cancer with an AUC value of 0.652. Finally, Gene set enrichment analysis showed that cell cycle-related pathways were significantly enriched in a high-risk group. RESULTS: Our results showed that glucose metabolism-related lncRNAs can affect breast cancer progression. 7 glucose metabolism-related lncRNAs prognostic signature was established to evaluate the OS of patients with breast cancer. PICSAR, LINC00839, AP001505.1, LINC00393 were risk factors and expressed highly in the high-risk group. A Nomogram was made based on this signature to judge patients' living conditions and prognosis. CONCLUSION: 7 glucose metabolism-related lncRNAs risk score model had a high prognostic value in breast cancer. PICSAR, LINC00839, AP001505.1, LINC00393 were risk factors. AP001505.1 expression was increased in most triple-negative breast cancer cells treated with high glucose, which may also take part in breast cancer progression and potential therapeutic targets.

Expression profile and prognostic values of SMC family members in HCC.

OBJECTIVE: The structural maintenance of chromosome (SMC) gene family, including 6 proteins, is involved in a wide range of biological functions in different human cancers. Nevertheless, there is little research on the expression patterns, potential functions and prognostic value of SMC genes in hepatocellular carcinoma (HCC). Based on publicly available databases and integrative bioinformatics analysis, we tried to determine the value of SMC gene expression in predicting the risk of developing HCC. METHODS: The expression and copy number variations data of SMC family members were obtained from TCGA (The Cancer Genome Atlas). We identified the prognostic values of SMC family members and their clinical features. GSEA (Gene Set Enrichment Analysis) was conducted to detect the mechanism underlying the involvement of SMC family members in liver cancer. We used Tumor Immune Estimation Resource database to explore the associations between TIICs (Tumor Immune Infiltrating Cells) and the SMC family members. RESULTS: Our analysis proved that downregulation of SMC family members was common modification in HCC patients. In HCC, the expression of SMC1A, SMC2, SMC3, SMC4, SMC6 were upregulated. Upregulation of SMC2, SMC3, and SMC4, along with the clinical stage of HCC, were associated with a poor prognosis according to the results of univariate and multivariate Cox proportional hazards regression analysis. SMC2, SMC3, and SMC4 are also related to tumor purity and immune infiltration levels of HCC. The GSEA results proved that SMC family members take part in numerous biological processes underlying tumorigenesis. CONCLUSION: In this study, we comprehensively analyzed the expression of SMC family members in patients with HCC. This can provide insights for further investigation of the SMC members as potential therapeutic targets in HCC and suggest that the use of SMC inhibitor targeting SMC2, SMC3, and SMC4 can be a practical strategy for the therapy of HCC.

Exosomal microRNAs shuttling between tumor cells and macrophages: cellular interactions and novel therapeutic strategies.

Extracellular vesicles secreted by tumor microenvironment (TME) cells are vital players in tumor progression through transferring nucleic acids and proteins. Macrophages are the main immune cells in TME and tumor associated macrophages (TAM) express M2 phenotype, which induce tumor proliferation, angiogenesis, invasion, metastasis and immune elimination, resulting in the subsequent evolution of malignancies. There are a high number of studies confirmed that tumor cells and TAM interact with each other through extracellular vesicles in various cancers, like pancreatic ductal adenocarcinoma, gastric cancer, breast cancer, ovarian cancer, colon cancer, glioblastoma, hepatocellular cancer, and lung cancer. Herein, this review summarizes the current knowledge on mechanisms of communications between tumor cells and TAM via extracellular vesicles, mainly about microRNAs, and targeting these events might represent a novel approach in the clinical implications of this knowledge into successful anti-cancer strategies.

Exchange-biased topological transverse thermoelectric effects in a Kagome ferrimagnet.

Kagome metal TbMn6Sn6 was recently discovered to be a ferrimagnetic topological Dirac material by scanning tunneling microscopy/spectroscopy measurements. Here, we report the observation of large anomalous Nernst effect and anomalous thermal Hall effect in this compound. The anomalous transverse transport is consistent with the Berry curvature contribution from the massive Dirac gaps in the 3D momentum space as demonstrated by our first-principles calculations. Furthermore, the transverse thermoelectric transport exhibits asymmetry with respect to the applied magnetic field, i.e., an exchange-bias behavior. Together, these features place TbMn6Sn6 as a promising system for the outstanding thermoelectric performance based on anomalous Nernst effect.

Malignant adenomyoepithelioma of the breast: cases report and literature review.

BACKGROUND: Malignant adenomyoepithelioma (MAME) of the breast is an extremely rare breast malignancy, in which they arise from either luminal epithelial or myoepithelial components, or both. At present, there is very little clinical data of MAME. CASE REPORT: We present two cases, one of them is a 34-year-old woman who underwent needle biopsy for a 3.2 cm-size mass in the right breast, and the pathology was MAME of breast. Another case is a 45-year-old woman who had a 3.0 cm-size mass in the right breast. We performed a breast-conserving surgery and sentinel lymph node biopsy, both of which were negative. The histopathology of these two cases was invasive carcinoma; however, these cases were eligible for MAME of the breast through combining with immunohistochemistry. CONCLUSIONS: MAME of the breast is very rare, and has a diverse cell morphology, which must be combined with immunohistochemistry to make a clear diagnosis. Besides, it should be differentiated from adenoid cystic cancer, malignant leafy tumor, spindle cell carcinoma, etc. The clinical characteristics and treatment strategies were further discussed in combination with the literature.

Anomalous Thermal Hall Effect in an Insulating van der Waals Magnet.

Two-dimensional (2D) van der Waals (vdW) magnets have been a fertile playground for the discovery and exploration of physical phenomena and new physics. In this Letter, we report the observation of an anomalous thermal Hall effect (THE) with κ_{xy}∼1×10^{-2} W K^{-1} m^{-1} in an insulating van der Waals ferromagnet VI_{3}. The thermal Hall signal persists in the absence of an external magnetic field and flips sign upon the switching of the magnetization. In combination with theoretical calculations, we show that VI_{3} exhibits a dual nature of the THE, i.e., dominated by topological magnons hosted by the ferromagnetic honeycomb lattice at higher temperatures and by phonons induced by the magnon-phonon coupling at lower temperatures. Our results not only position VI_{3} as the first ferromagnetic system to investigate both anomalous magnon and phonon THEs, but also render it as a potential platform for spintronics-magnonics applications.

Tumor-derived exosomal circPSMA1 facilitates the tumorigenesis, metastasis, and migration in triple-negative breast cancer (TNBC) through miR-637/Akt1/ß-catenin (cyclin D1) axis.

Circular RNAs (circRNAs) are increasingly gaining importance and attention due to their diverse potential functions and their value as diagnostic biomarkers (disease specific). This study aims to explore the novel mechanisms by which exosome-contained circRNAs promote tumor development and metastasis in TNBC. We identified increased circRNA circPSMA1 in TNBC cells, their exosomes, and serum exosomes samples from TNBC patients. The overexpression of circPSMA1 promoted TNBC cell proliferation, migration, and metastasis both in vitro and in vivo. Moreover, we investigated the tumor-infiltrating immune cells (TICs) or stromal components in immune microenvironment (IME), and identified the significant differences in the immune cells between TNBC and non-TNBC samples. Mechanistically, circPSMA1 acted as a "miRNAs sponge" to absorb miR-637; miR-637 inhibited TNBC cell migration and metastasis by directly targeted Akt1, which recognized as a key immune-related gene and affected downstream genes ß-catenin and cyclin D1. Subsequent co-culture experiments also demonstrated that exosomes from TNBC carrying large amounts of circPSMA1 could transmit migration and proliferation capacity to recipient cells. Kaplan-Meier plots showed that high expression of Akt1 and low expression of mir-637 are highly correlated with poor prognosis in patients with lymph node metastasis of TNBC. Collectively, all these results reveal that circPSMA1 functions as a tumor promoter through the circPSMA1/miR-637/Akt1-ß-catenin (cyclin D1) regulatory axis, which can facilitate the tumorigenesis, metastasis, and immunosuppression of TNBC. Our research proposes a fresh perspective on novel potential biomarkers and immune treatment strategies for TNBC.

The role of long non-coding RNAs in drug resistance of cancer.

Long non-coding RNAs (lncRNAs), a class of long RNAs, are longer than 200 nucleotides in length but lack protein-coding capacity. LncRNAs, as critical genomic regulators, are involved in genomic imprinting regulation, histone modification and gene expression regulation as well as tumor initiation and progression. However, it is also found that lncRNAs are associated with drug resistance in several types of cancer. Drug resistance is an important reason for clinical chemotherapy failure, and the molecular mechanism of tumor resistance is complex, which is a process of multi-cause, multi-gene and multi-signal transduction pathway interaction. Then comprehending the mechanisms of chemoresistance will help find ways to control the tumor progression effectively. Therefore, in this review, we will construct lncRNAs /drug resistance interaction network and shed light on the role of lncRNAs in drug resistance.

Identification and validation of tumor microenvironment-related prognostic biomarkers in breast cancer.

BACKGROUND: Stromal cells and immune cells in tumor microenvironment (TME) have been reported to have significant value in the diagnosis and prognosis of cancers. We aimed to identify key biomarkers predicting survival in the TME of breast cancer. METHODS: Cell type enrichment analysis was performed to estimate cell scores using the xCell method with gene expression data from public database. Least absolute shrinkage and selection operator (LASSO) regression was used to identify key signature from the cell scores. RESULTS: Totally, 50 cells in TME had different scores between 1,078 breast cancer tissues and 112 adjacent normal tissues. We identified a 4-cell signature predicting breast cancer survival, including myocytes, natural killer T cell (NKT), conventional dendritic cell (cDC) and sebocytes, which was validated in the test set. Further analysis showed that cDC score was a key signature predicting prognosis of breast cancer. cDC score was significantly associated with molecular classification and stage of breast cancer, as well as expression level of Ki67. Spearman's correlation analysis found that cDC score was inversely correlated with the expression level of HER2. High cDC score may predicate better pathological complete response rate. Mechanism analysis indicated high cDC score was associated with elevated immune activity; IL-2 was a key gene associated with high cDC score; and Breast cancer patients with high IL-2 expression had a longer survival time. CONCLUSIONS: In conclusion, cDC score was a key signature predicting prognosis for breast cancer. cDCs may exert antitumor effects by upregulating IL-2.

Identification of circRNA-miRNA networks for exploring an underlying prognosis strategy for breast cancer.

Aim: Circular RNAs (circRNAs) still have many potential functions in the process of tumor development that are not completely understood. The study aims to explore novel circRNAs and their mechanisms of action in breast cancer (BCa). Materials & methods: A combination strategy of RNA-sequencing (RNA-seq) technique, quantitative real-time PCR and bioinformatic analysis was employed to identify the potential mechanisms involving differentially expressed circRNAs in the serum exosomes and tissues of BCa patients. Results: The expression levels of hsa-circRNA-0005795 and hsa-circRNA-0088088 were significantly different both in serum exosomes and tissues and might function as competing endogenous RNAs and play vital roles in BCa development. Conclusion: We constructed two circRNA-miRNA networks and provided new insight into the prognosis and therapy of BCa using circRNAs from serum exosomes.

Circular RNA circVAPA regulates breast cancer cell migration and invasion via sponging miR-130a-5p.

Aim: We aimed to explore the roles of circular RNA, circVAPA in regulating cell migration and invasion of breast cancer. Materials & methods: CircVAPA expression was detected in breast cancer tissues and cells. The role of circVAPA was evaluated by MTT assay, wound-healing and transwell assay. The relationship between circVAPA and miR-130a-5p and the location of circVAPA were explored. Results: We discovered that circVAPA was dysregulated in breast cancer tissues and cells. Ectopic circVAPA regulated breast cancer migration, invasion and proliferation. CircVAPA was mainly expressed in the cytoplasm and could act as a miRNA sponge for miR-130a-5p, but did not regulate its parental gene. Conclusion: CircVAPA may promote migration and invasion capacity of breast cancer via harboring miR-130a-5p.

Microenvironment-induced TIMP2 loss by cancer-secreted exosomal miR-4443 promotes liver metastasis of breast cancer.

We aimed to investigate the role of exosomal miR-4443 in metastasis of breast cancer (BCa). In vitro wound-healing assay and transwell invasion assay were used to investigate effect of miR-4443 on BCa cells. Animal experiments were performed to confirm its effects in vivo. miR-4443 promotes the metastasis of BCa cells through downregulating tissue inhibitors of metalloproteinase 2 (TIMP2) and upregulating matrix metalloproteinases (MMPs). Highly invasive BCa cells have a higher expression of miR-4443 in both cells and exosomes. The exosomes derived from highly invasive BCa cells mainly gather in the primary tumor and liver. In vivo, overexpression of miR-4443 in noninvasive BCa cells induces liver metastasis, accompanied with downregulated TIMP2, and upregulated MMP-2 in both the primary tumor and liver. When we armed MCF-10A exosomes with miR-4443 inhibitors to treat mice bearing high-miR-4443 tumors, exosomes accumulated in the primary tumor, and liver following the upregulation of TIMP2 and downregulation of MMP2, and the metastasis was inhibited. Highly invasive BCa cells destroy natural barriers against metastasis by delivering exosomal miR-4443 to stromal cells of the primary tumor and impairing TIMP2, consequently activating MMP; circulating exosomal miR-4443 might promote BCa cells lodging in future metastatic sites through the similar mechanisms.

The emerging role of circular RNAs in breast cancer.

Breast cancer (BCa) is one of the most frequently diagnosed cancers and leading cause of cancer deaths among females worldwide. Circular RNAs (circRNAs) are a new class of endogenous regulatory RNAs characterized by circular shape resulting from covalently closed continuous loops that are capable of regulating gene expression at transcription or post-transcription levels. With the unique structures, circRNAs are resistant to exonuclease RNase R and maintain stability more easily than linear RNAs. Recently, an increasing number of circRNAs are discovered and reported to show different expression in BCa and these dysregulated circRNAs were correlated with patients' clinical characteristics and grade in the progression of BCa. CircRNAs participate in the bioprocesses of carcinogenesis of BCa, including cell proliferation, apoptosis, cell cycle, tumorigenesis, vascularization, cell invasion, migration as well as metastasis. Here we concentrated on biogenesis and function of circRNAs, summarized their implications in BCa and discussed their potential as diagnostic and therapeutic targets for BCa.

Circular RNA circASS1 is downregulated in breast cancer cells MDA-MB-231 and suppressed invasion and migration.

AIM: The study aimed to investigate the role of circular RNA circASS1 in breast cancer cells. MATERIALS & METHODS: Circular RNAs microarray expression profile were analyzed in MCF-7, MDA-MB-231, and qRT-PCR and western blotting were used to quantify expression of circASS1 and its parental gene ASS1. Wound healing, migration and invasion assay were performed. Luciferase assay system was used to detect harbored miRNA. RESULTS: CircASS1 in MDA-MB-231 is downregulated comparing to MCF-7, and overexpression of circASS1 could suppress invasion and migration. While silence, it could promote invasion and migration. MiR-4443 functioning as a tumor promoter gene could be captured by circASS1. ASS1 is upregulated in loss-of-function experiments, while downregulated in gain-of-function experiments. CONCLUSION: CircASS1 suppresses invasion and migration capacity of breast cancer cells and harbored miR-4443.

Exosome: a novel mediator in drug resistance of cancer cells.

Exosomes are small membrane vesicles with a diameter of 40-100 nm, which are released into the intracellular environment. Exosomes could influence the genetic and epigenetic changes of receptor cells by promoting the horizontal transfer of various proteins or RNAs, especially miRNAs. Moreover, exosomes also play an important role in tumor microenvironment. Exosomes could promote the short- and long-distance exchanges of genetic information by acting as mediators of cell-to-cell communication. In addition, exosomes participate in drug resistance of tumor cells by genetic exchange between cells. It is reported that exosomes could be absorbed by recipient cells and transmit chemoresistance from drug-resistant tumor cells to sensitive ones. Then understanding the mechanisms of chemotherapy failure and controlling tumor progression effectively will be a major challenge for us. Therefore, in this review, we will briefly reveal the role of exosomes in drug resistance.

The regulatory roles of lncRNAs in the process of breast cancer invasion and metastasis.

Breast cancer (BC) is the most common cancer and principal cause of death among females worldwide. Invasion and metastasis are major causes which influence the survival and prognosis of BC. Therefore, to understand the molecule mechanism underlying invasion and metastasis is paramount for developing strategies to improve survival and prognosis in BC patients. Recent studies have reported that long non-coding RNAs (lncRNAs) play critical roles in the regulation of BC invasion and metastasis through a variety of molecule mechanisms that endow cells with an aggressive phenotype. In this article, we focused on the function of lncRNAs on BC invasion and metastasis through participating in epithelial-to-mesenchymal transition, strengthening cancer stem cells generation, serving as competing endogenous lncRNAs, influencing multiple signaling pathways as well as regulating expressions of invasion-metastasis related factors, including cells adhesion molecules, extracellular matrix, and matrix metallo-proteinases. The published work described has provided a better understanding of the mechanisms underpinning the contribution of lncRNAs to BC invasion and metastasis, which may lay the foundation for the development of new strategies to prevent BC invasion and metastasis.

Circular RNA hsa_circ_0052112 promotes cell migration and invasion by acting as sponge for miR-125a-5p in breast cancer.

OBJECTIVES: Accumulating evidence has been reported that circular RNAs (circRNAs) are a class of relatively stable, non-coding RNAs, which are involved in the progression of many types of diseases. However, the mechanism of hsa_circ_0052112 in breast cancer cells is not entirely clear. Hsa_circ_0052112, generated from the ZNF83 gene, is selected by analyzing circRNA expression profiles of breast cancer cell by using microarray assay. In this study, we will show the role of hsa_circ_0052112 in regulating cell invasion and migration in breast cancer. METHODS: The expression level of hsa_circ_0052112 in MCF-7 and MDA-MB-231 cells was detected by RT-qPCR; we performed transwell assay to evaluate breast cancer cells' migration and invasion; predicated circRNA/miRNAs interaction using the miRanda and RNAhybrid software; identified the relationship between hsa_circ_0052112 and miR-125a-5p by luciferase activity assay and show the localization of hsa_circ_0052112 by FISH assay and show the significance of ZNF83 in clinical prognosis by Kaplan-Meier survival analysis. RESULTS: Hsa_circ_0052112 expression was significantly higher in MDA-MB-231 cells than that in MCF-7 cells. Overexpression of hsa_circ_0052112 promoted cell migration and invasion in breast cancer. Inversely, down-regulation of hsa_circ_0052112 suppressed breast cancer cells migration and invasion. Hsa_circ_0052112 was mostly located in cytoplasm. Hsa_circ_0052112 could directly sponge to miR-125a-5p; overexpression of miR-125a-5p significantly inhibited breast cancer cells migration and invasion. However, high or low expression of miR-125a-5p was not correlated with relapse free survival (RFS) by TCGA database validation, but high expression of ZNF83 was closely correlated with poor RFS by Kaplan-Meier plotter. CONCLUSIONS: These data suggest that hsa_circ_0052112 may be a potent biomarker for breast cancer, and may provide a new perspective on treatment of breast cancer.

Circular RNA hsa_circ_0072995 promotes breast cancer cell migration and invasion through sponge for miR-30c-2-3p.

AIM: To study the role of hsa_circ_0072995 in regulating the invasion and migration of breast cancer cells. MATERIALS & METHODS: Hsa_circ_0072995 expression was confirmed by quantitative real-time PCR; evaluating the migration and invasion of breast cancer cells through transwell assay; predicating circRNA/microRNAs interaction using the miRanda and RNAhybrid software; identifying the relationship between hsa_circ_0072995 and miR-30c-2-3p by luciferase activity assay; detecting the location of hsa_circ_0072995 by Fluorescence in situ hybridization assay. RESULTS: Hsa_circ_0072995 was significantly upregulated in MDA-MB-231 cells compared with MCF-7 cells. Hsa_circ_0072995 regulated the invasion and migration of breast cancer cells. Hsa_circ_0072995 existed in the nucleus and cytoplasm, and the proportion of the two was roughly equal. Hsa_circ_0072995 bound to miR-30c-2-3p. Overexpression of miR-30c-2-3p inhibited breast cancer cells migration and invasion. Low expression of miR-30c-2-3p was correlated with poor overall survival by The Cancer Genome Atlas database. CONCLUSION: Hsa_circ_0072995 may be a novel biomarker for breast cancer, and may function in metastasis of breast cancer.